Use of acidic glycoprotein ORM for the preparation of anti-aging drugs

Abstract

This invention belongs to the field of biomedicine, specifically relating to the application of acidic glycoprotein ORM in the preparation of anti-aging drugs. This invention discovers that ORM protein is closely related to the occurrence and development of aging. In cell models, exogenous ORM can significantly improve D-galactose and eptoposide-induced cell senescence and angiotensin II-induced cell senescence. In animal models, overexpression of ORM1 in cardiomyocytes can significantly improve diastolic dysfunction in aged mice. In addition, since acidic glycoprotein ORM is an endogenous protein, it has high safety as a drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of acidic glycoprotein ORM in the preparation of anti-aging drugs. Background Technology

[0002] Aging is a gradual and irreversible pathophysiological process. It manifests as a decline in tissue and cellular function, and a significantly increased risk of various age-related diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic diseases, musculoskeletal diseases, and immune system diseases. Although advancements in modern medicine have promoted human health and greatly extended life expectancy, with the aging of society, various chronic diseases are gradually becoming the most important cause of disability and death among the elderly. The aging of the body and organs is essentially cellular aging. Cellular aging is a necessary stage in the growth and development of cells in the human body, involving the degeneration of multiple cellular functions and structures. Cellular aging can be divided into two categories: replicative aging and stress-induced premature aging. Replicative aging refers to premature aging that occurs after a limited number of cell divisions and the gradual shortening of telomeres at the ends of chromosomes. This leads to the arrest of cell proliferation and loss of differentiation capacity. Stress-induced premature aging refers to premature aging that occurs in response to pathological stimuli (such as DNA damage and oxidative stress). Regardless of the type of aging, senescent cells will exhibit characteristics such as increased particle size, increased size, elevated expression levels of the aging-related factor β-galactosidase, and increased levels of cell cycle-dependent kinase inhibitors and cell cycle repressor proteins such as p53, p21, and p16.

[0003] Currently, the mechanisms of cellular senescence and the pathways through which it leads to cellular dysfunction are not fully understood. Delaying cellular and tissue functional damage and alterations, inhibiting aging-related signaling, and discovering potential anti-aging drugs are major problems that need to be addressed. With the aging population and increasing life expectancy, exploring the potential mechanisms of aging and cellular senescence, and investigating the potential anti-cellular senescence effects of candidate drugs, has become particularly important. Studying the mechanisms of cellular senescence helps to further clarify the development direction of anti-aging drugs and has high research and application value.

[0004] α-1 acid glycoprotein (AGP), or orosomucoid, is an acute-phase protein in the liver. Stress stimuli such as trauma, bacterial infection, and inflammation can induce a significant increase in ORM levels in the liver and serum. Human ORM has two subtypes (ORM1 and ORM2), while mouse ORM has three (ORM1, ORM2, and ORM3). ORM1 is the predominant subtype in peripheral tissues. However, the role of ORM in anti-aging processes is currently lacking in research. Summary of the Invention

[0005] The purpose of this invention is to provide an application of acidic glycoprotein ORM in the preparation of anti-aging drugs, and to discover that exogenous ORM can significantly improve D-galactose and epostioside-induced cell senescence and angiotensin II-induced cell senescence, providing a new approach for the development of anti-aging drugs.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides an application of an acidic glycoprotein ORM in the preparation of products for treating or slowing down aging; Furthermore, the product includes pharmaceuticals.

[0007] Furthermore, the dosage forms of the drug include tablets, capsules, oral preparations, and injections.

[0008] Furthermore, the product slows down aging by improving D-galactose, eptoposide, and angiotensin II-induced cellular senescence.

[0009] Furthermore, the improvement of D-galactose, eptoposide, and angiotensin II-induced cellular senescence is achieved by reducing the expression levels of senescence marker proteins P53 and P21.

[0010] Furthermore, the product slows down aging by improving diastolic dysfunction in the heart of aged mice.

[0011] Furthermore, the coding sequence of the acidic glycoprotein ORM is shown in SEQ ID NO.1.

[0012] The present invention also provides a product for treating or slowing down aging, the product comprising the aforementioned acidic glycoprotein ORM or acidic glycoprotein ORM promoter.

[0013] Furthermore, the acidic glycoprotein ORM promoter is an overexpression vector of the acidic glycoprotein ORM coding sequence that can increase the expression level of acidic glycoprotein ORM.

[0014] Furthermore, the product also includes pharmaceutically or immunologically acceptable carriers or excipients.

[0015] Beneficial effects: This invention reveals that ORM proteins are closely related to the occurrence and development of aging. In cell models, exogenous ORMs significantly improve D-galactose- and eptoposide-induced cellular senescence, as well as angiotensin II-induced cellular senescence. In animal models, overexpression of ORM1 in cardiomyocytes significantly improves diastolic dysfunction in aged mice. This invention provides a novel use for acidic glycoprotein ORMs in the preparation of drugs to slow aging. Because acidic glycoprotein ORMs are endogenous proteins, they have a high safety profile as pharmaceuticals. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The diagram shows the effect of exogenous ORM on D-galactose-induced senescent positive HL-1 cells. In the diagram, A is the staining diagram and B is the comparison diagram of the number of β-galactosidase-positive cells. Figure 2 The image shows the effect of exogenous ORM on senescent positive HL-1 cells of Etoposide. In the image, A is the staining image and B is a comparison of the number of senescent positive HL-1 cells of Etoposide. Figure 3 The figure shows the effect of exogenous ORM on the expression of senescence marker proteins in HL-1 cells. In the figure, A shows the effect of exogenous administration of 10 μg / mL ORM on the mRNA level of senescence marker protein P53, B shows the effect of exogenous administration of 10 μg / mL ORM on the mRNA level of senescence marker protein P21, C shows the effect of exogenous administration of 40 μg / mL ORM on the mRNA level of senescence marker protein P21, and D shows the effect of exogenous administration of 40 μg / mL ORM on the protein level of senescence marker protein P21. Figure 4 The image shows the effect of exogenous ORM on angiotensin II-induced senescent positive HUVEC cells. In the image, A is the staining image, and B is a comparison of the number of angiotensin II-induced senescent β-galactosidase-stained positive HUVEC cells. Figure 5 The figure shows the effect of exogenous ORM on the expression of senescence marker proteins in HUVEC cells. In the figure, A represents the effect of exogenous administration of 20 μg / mL ORM on the mRNA level of senescence marker protein P53, and B represents the effect of exogenous administration of 20 μg / mL ORM on the mRNA level of senescence marker protein P21. Figure 6 The image shows the effect of myocardial overexpression of ORM1 on ventricular remodeling in aged mice. In the image, A is a comparison of the hearts of mice under different treatments, and B is the tibia ratio of the hearts of mice under different treatments. Figure 7The figure shows the effect of myocardial overexpression of ORM1 on diastolic function in aged mice. In the figure, A represents echocardiography of mice in different treatment groups, B represents EF value of mice in different treatment groups, C represents FS value of mice in different treatment groups, D represents EDV value of mice in different treatment groups, and E represents LVIDd value of mice in different treatment groups. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1: Exogenous ORM improves HL-1 senescence in cardiomyocytes 1.1 Experimental Materials HL-1 mouse cardiomyocytes (Wuhan Pronosei Life Sciences Co., Ltd.), 6-well cell culture plates (Corning), microscope. Main reagents: ORM (Sigma), D-galactose (Shanghai Sangon Biotech Co., Ltd.), Etoposide (Beyotime), cell senescence β-galactosidase staining kit (Beyotime).

[0024] 1.2 Cell grouping and experimental treatment Two in vitro induced cell senescence models were used: (1) D-galactose-induced cell senescence: HL-1 cells were seeded in 6-well cell culture plates and divided into control group, D-galactose group, ORM group, and D-galactose + ORM group, with 3 wells in each group. After the HL-1 cells reached a confluence density of 60%, the culture medium was changed. The D-galactose group was replaced with complete culture medium containing 10 mg / mL D-galactose, the ORM group was replaced with complete culture medium containing 10 μg / mL D-galactose, the D-galactose + ORM group was replaced with complete culture medium containing both the above concentrations of D-galactose and ORM, and the control group was replaced with complete culture medium containing an equal volume of 1×PBS. The cells were incubated in a cell incubator for 72 h.

[0025] (2) Etoposide-induced cell senescence: HL-1 cells were seeded in 6-well cell culture plates and divided into control group, Etoposide group, ORM group, and Etoposide+ORM group, with 3 wells in each group. After the HL-1 cells reached a confluence density of 60%, the culture medium was changed. The Etoposide group was replaced with complete culture medium containing 5 μM Etoposide, the ORM group was replaced with complete culture medium containing 40 μg / mL Etoposide, the Etoposide+ORM group was replaced with complete culture medium containing both the above concentrations of Etoposide and ORM, and the control group was replaced with complete culture medium containing an equal volume of 1×PBS. The cells were incubated in a cell incubator for 24 h.

[0026] 1.3 Cellular senescence β-galactosidase staining This step uses a cell senescence β-galactosidase staining kit (Beyotime) for staining, and specifically includes the following steps: (1) Remove the cell culture medium, wash once with PBS, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 minutes.

[0027] (2) Remove the cell fixative and wash the cells three times with PBS for three minutes each time.

[0028] (3) Remove the PBS and add 1 mL of staining working solution to each blank. The staining working solution is prepared as follows: staining solution A (10 μL), staining solution B (10 μL), staining solution C (930 μL), X-Gal solution (50 μL).

[0029] (4) Incubate overnight at 37°C and observe under a regular optical microscope.

[0030] 1.4 Real-time quantitative PCR Total RNA was extracted from cells and reverse transcribed using the Takara PrimeScript™ RT Master Mix kit. Real-time quantitative PCR was used to detect the target genes in the cDNA samples obtained from the reverse transcription. -△△Ct The relative mRNA expression was calculated using a method with GAPDH as an internal reference for normalization. Primer sequence information is as follows: GAPDH (F: TGTGTCCGTCGTGGATCTGA; R: TTGCTGTTGAAGTCGCAGGAG); p53 (F: CCCCTGTCATCTTTTGTCCCT; R: AGCTGGCAGAATAGCTTATTGAG); p21 (F: GCAAAGTGTGCCGTTGTCTC; R: AAAGTTCCACCGTTCTCGGG).

[0031] 1.5 Immunoblotting Total protein was extracted from cells, and protein concentration was determined using the BCA method. After denaturation, protein samples were subjected to electrophoresis on an SDS-PAGE gel, transferred to a membrane, and immunoblotted. The primary antibodies used were anti-rabbit p21 (1:1000, Proteintech) and anti-rabbit GAPDH (1:10000, Proteintech), and the secondary antibody was goat anti-rabbit IgG (1:3000, CST).

[0032] 1.6 Statistical Analysis All values ​​are expressed as mean ± SEM. One-way ANOVA was used for pairwise comparisons of two or more groups, and two-way ANOVA was used for two-way comparisons between groups. Graphpad Prism-8 statistical software (La Jolla, CA) was used for data analysis. P < 0.05 was considered statistically significant, where * indicates P < 0.05; ** indicates P < 0.01; and **** indicates P < 0.0001.

[0033] 1.7 Experimental Results (1) Exogenous ORM reduces senescent β-galactosidase-positive HL-1 cells Depend on Figure 1 , Figure 2It is evident that exogenous administration of 10 μg / mL ORM can significantly reduce the number of D-galactose-induced senescent β-galactosidase-positive HL-1 cells, and exogenous administration of 40 μg / mL ORM can significantly reduce the number of Etoposide-induced β-galactosidase-positive cells.

[0034] (2) Exogenous ORM reduces the expression of senescence marker proteins in HL-1 cells. Depend on Figure 3 It is evident that exogenous administration of 10 μg / mL ORM significantly reduced the mRNA levels of D-galactose-induced aging marker proteins P53 and P21 (A, B), and exogenous administration of 40 μg / mL ORM significantly reduced the mRNA and protein levels of Etoposide-induced aging marker protein P21 (C, D).

[0035] Example 2: Exogenous ORM improves senescence of endothelial cells HUVEC 2.1 Experimental Materials HUVEC human umbilical vein endothelial cells (Wuhan Pronosei Life Sciences Co., Ltd.), 6-well cell culture plates (Corning), microscope. Main reagents: ORM (Sigma), angiotensin II (MCE), cell senescence β-galactosidase staining kit (Beyotime).

[0036] 2.2 Cell grouping and experimental treatment An angiotensin II (Ang II)-induced cell senescence model was used: HUVEC cells were seeded in 6-well cell culture plates and divided into control, Ang II, ORM, and Ang II+ORM groups, with 3 wells in each group. After the HUVEC cells reached 60% confluence, the culture medium was changed. The Ang II group was replaced with complete medium containing 10 nM Ang II, the ORM group with complete medium containing 20 μg / mL Ang II, the Ang II+ORM group with complete medium containing both the above concentrations of Ang II and ORM, and the control group with complete medium containing an equal volume of 1×PBS. Cells were incubated for 48 h.

[0037] 2.3 Cell senescence β-galactosidase staining This step uses a cell senescence β-galactosidase staining kit (Beyotime) for staining, and specifically includes the following steps: (1) Remove the cell culture medium, wash once with PBS, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 minutes.

[0038] (2) Remove the cell fixative and wash the cells three times with PBS for three minutes each time.

[0039] (3) Remove the PBS and add 1 mL of staining working solution to each blank. The staining working solution is prepared as follows: staining solution A (10 μL), staining solution B (10 μL), staining solution C (930 μL), X-Gal solution (50 μL).

[0040] (4) Incubate overnight at 37°C and observe under a regular optical microscope.

[0041] 2.4 Real-time quantitative PCR Total RNA was extracted from cells and reverse transcribed using the Takara PrimeScript™ RT Master Mix kit. Real-time quantitative PCR was used to detect the target genes in the cDNA samples obtained from the reverse transcription. -△△Ct The relative mRNA expression was calculated using a method with GAPDH as an internal reference for normalization. Primer sequence information is as follows: GAPDH (F: AAAGCCTGCCGGTGACTAAC; R: AGGAAAAGCATCACCCGGAG); p53 (F: AGAAAACCTACCAGGGCAGC; R: ACATCTTGTTGAGGGCAGGG); p21 (F: AGTCAGTTCCTTGTGGAGCC; R: GCATGGGTTCTGACGGACAT).

[0042] 2.5 Statistical Analysis All values ​​are expressed as mean ± SEM. One-way ANOVA was used for pairwise comparisons of two or more groups. Graphpad Prism-8 statistical software (La Jolla, CA) was used for data analysis. P < 0.05 was considered statistically significant, where ** indicates P < 0.01; **** indicates P < 0.0001.

[0043] 2.6 Experimental Results (1) Exogenous ORM reduces senescent β-galactosidase-positive HUVEC cells Depend on Figure 4 It is evident that exogenous administration of 20 μg / mL ORM can significantly reduce the number of angiotensin II-induced senescent β-galactosidase-positive HUVEC cells.

[0044] (2) Exogenous ORM reduces the expression of senescence marker proteins in HUVEC cells. Depend on Figure 5 It is evident that exogenous administration of 20 μg / mL ORM can significantly reduce the mRNA levels of angiotensin II-induced aging marker proteins P53 and P21.

[0045] Example 3: Cardiac cell-specific overexpression of ORM1 improves diastolic dysfunction in aged rats. 3.1 Experimental Materials C57BL / 6J mice. AAV-cTNT-OE-NC / ORM1 adeno-associated virus (Hanheng Biotechnology).

[0046] 3.2 Animal grouping and experimental treatment Eighteen-month-old C57BL / 6J mice were randomly assigned using a random number table to a control group and an ORM1 overexpression group, with 3-6 mice in each group. The mice were administered myocardial-specific overexpression of adeno-associated virus AAV-cTNT-OE-NC / ORM1 via tail vein injection. Two months later, the mice were tested and tissue samples were collected.

[0047] 3.3 Echocardiography Cardiac function in mice was assessed using a two-dimensional and M-mode ultrasound diagnostic instrument (Esaote MyLab One / Touch, Italy). Mice were anesthetized with isoflurane gas, and depilatory cream was applied to their chests. After 5 minutes, the chest hair was removed with a dry cotton ball. Ultrasound lubricant was applied, and the mice were examined using an SL3116 probe at a frequency of 22 MHz.

[0048] 3.4 Statistical Analysis All values ​​are expressed as mean ± SEM. One-way ANOVA was used for pairwise comparisons of two or more groups. Graphpad Prism-8 statistical software (La Jolla, CA) was used for data analysis. P < 0.05 was considered statistically significant, where ** indicates P < 0.01; **** indicates P < 0.0001.

[0049] 3.5 Experimental Results (1) Myocardial overexpression of ORM1 reduces ventricular remodeling in aged mice Depend on Figure 6 It is evident that, compared with the hearts of 5-month-old mice, the hearts of 20-month-old mice showed increased volume and tibia-to-heart ratio, indicating ventricular remodeling in the hearts of older mice. However, after overexpression of ORM1 in the myocardium of older mice, the heart volume decreased and the tibia-to-heart ratio decreased, suggesting that ventricular remodeling was reduced.

[0050] (2) Overexpression of ORM1 in myocardium reduces diastolic dysfunction in aged mice. Depend on Figure 7It is evident that, compared with the hearts of 5-month-old young mice, the EF and FS values ​​of the hearts of 20-month-old aged mice remained unchanged, while the EDV and LVIDd values ​​were significantly increased, indicating the presence of cardiac diastolic dysfunction without change in cardiac function. After myocardial overexpression of ORM1, the EDV and LVIDd values ​​returned to the youthful state, indicating a reduction in cardiac diastolic dysfunction.

[0051] Based on the above experimental results, the ORM protein of this invention is closely related to the occurrence and development of aging. In cell models, exogenous ORM can significantly improve the aging phenotype of cardiomyocytes and endothelial cells. In animal models, overexpression of ORM1 in cardiomyocytes can significantly improve diastolic dysfunction in aged mice.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of acidic glycoprotein ORM1 in the preparation of products for treating or slowing down aging, characterized in that, The product is a medicine.

2. The application as described in claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, and injections.

3. The application as described in claim 1, characterized in that, The product slows down aging by improving D-galactose, eptoposide, and angiotensin II-induced cellular senescence.

4. The application as described in claim 3, characterized in that, The improvement of D-galactose, eptoposide, and angiotensin II-induced cellular senescence is achieved by reducing the expression levels of senescence marker proteins P53 and P21.

5. The application as described in claim 1, characterized in that, The product slows down aging by improving diastolic dysfunction in the heart of aged mice.

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